UDP-N-acetylgalactosamine:globoside alpha-3-N-acetylgalactosaminyltransferase. Purification, characterization, and some properties.
A UDP-N-acetylgalactosamine:globoside alpha-3-N-acetylgalactosaminyltransferase has been purified over 3500-fold in 4% yield from a Triton X-100 extract of canine spleen microsomes by affinity chromatography on globoside acid-agarose. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme revealed two major bands with molecular weights of 66,000 and 56,000. Judging from the molecular weight of 120,000 estimated by Sephadex gel filtration in Triton X-100 and the above electrophoretic result, the enzyme presumably exists normally as a dimer. It required Mn2+ for its activity and had a pH optimum at 6.7-6.9. The enzyme catalyzes the transfer of N-acetylgalactosamine in alpha 1 leads to 3 linkage to globoside. Neither Fuc alpha 1 leads to 2Gal beta 1 leads to 4Glc nor H blood group substance nor deglycosylated mucin were acceptors. This indicates that the enzyme was distinct from both the N-acetylgalactosaminyltransferases converting the H to the A blood group substance and catalyzing synthesis of the Ser(Thr)-GalNAc linkage. Studies on substrate specificities indicate that the preferred substrates have the general structure GalNAc beta 1 leads to 3Gal-OR in which the nature of the R moiety has relatively little effect on activity. Kinetic analysis indicates UDP is a competitive inhibitor with respect to UDP-N-acetylgalactosamine and a noncompetitive inhibitor with respect to globoside. These studies demonstrate the first report of the properties of a purified enzyme catalyzing the transfer of sugar residues to glycolipids.
D000818:Animals; D002413:Cations, Divalent; D004285:Dogs; D004355:Drug Stability; D005700:Galactosyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D005779:Immunodiffusion; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D008970:Molecular Weight; D017350:N-Acetylgalactosaminyltransferases; D013154:Spleen / Q000201:enzymology*; D013379:Substrate Specificity
D002413:Cations, Divalent; D005700:Galactosyltransferases; D017350:N-Acetylgalactosaminyltransferases; C023216:globoside alpha-N-acetylgalactosaminyltransferase